Significance: Effective delivery of nucleic acid therapeutics (NAT) is necessary for the treatment and/or prevention of a myriad of genetic diseases such as Epidermolysis bullosa (EB), as well as for other non-genetic diseases, including infectious diseases, cancers, and slow-healing wounds. While advancements have been made in the NAT field, effective NAT delivery remains the bottleneck for gene therapy. There are currently no FDA-approved methods for gene delivery in humans. Epidermolysis Bullosa (EB) is a rare and serious group of skin disorders characterized by painful blistering of skin in response to heat, pressure or friction (from rubbing), and minor injuries. Recessive Dystrophic Epidermolysis Bullosa (RDEB) is an optimal use case to demonstrate direct trans-tissue delivery of NATs to treat genetic dermal diseases but there are currently no local and painless delivery approaches. NATs for RDEB cannot be administered via site-specific injections, since only the cells immediately surrounding the injection site and exposed to the carrier will receive the therapeutic. They also cannot be delivered topically as it is extremely painful, and the NATs are unable to permeate across several layers of cells to reach the basal epidermal layer where the dysfunctional collagen that causes EB is made. Hypothesis: We hypothesize that our patented, portable, and painless system in which stable sub-micron therapeutic-loaded droplets are generated in a transitional flow regime can be used to deliver NATs into the basal dermal layer via low-pressure transdermal application, thus having great impact in treating EB and other diseases. Preliminary Data: Droplette has been tested extensively across ex vivo, in vivo, and pilot human studies. We have demonstrated that Droplette can deliver naked large (>1 MDa) mRNA to the basal layer of mucosal tissue. We have delivered GFP plasmid in H9c2 primary cardiomyocyte monolayers, and in cells 5-8 layers deep in in vitro tissue models (EpiOcular?, Mattek Corp). In pilot human studies, we have shown that Droplette provides up to 2X more hydration in skin which sustains over a longer period of time (2h) compared to topical application while being painless and low-force. Specific Aims: We will optimize and demonstrate the efficacy of Droplette in delivering NATs. In Specific Aim 1, we will complete parametric optimization of the Droplette device and NAT packaging. We will demonstrate uniform penetration and optimize tunable parameters (e.g. droplet size, velocity) for NAT delivery. We will explore the use of three packaging systems (Naked NATs, Lipid nanoparticles, Adeno-Associated Viruses), to deliver NATs (DNA, RNA) into a variety of cells and tissues (3D cell aggregates, organoids, ex vivo epithelial tissue). In Specific Aim 2, we will perform preclinical studies in a mouse model for EB to assess the efficacy of our delivery system to achieve delivery of (1) NanoLuciferase constructs into mouse dermal tissue and (2) Lamc2 gene into the basal layer of dermal tissue of the EB mouse model B6.Cg-Lamc2tm1Uit/DcrJ to resolve lesions. Collectively, results from these studies will demonstrate the efficacy of our device for trans-tissue NAT delivery.